Annexin A2 Is a C-terminal PCSK9-binding Protein That Regulates Endogenous Low Density Lipoprotein Receptor Levels
Annexin A2 Is a C-terminal PCSK9-binding Protein That Regulates Endogenous Low Density Lipoprotein Receptor Levels
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DOI：
10.1074/jbc.m805971200
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发表时间：
2008-11-14
影响因子：
4.8
通讯作者：
Seidah, Nabil G.
中科院分区：
文献类型：
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作者：
Mayer, Gaetan;Poirier, Steve;Seidah, Nabil G.
前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK 9），促进肝脏低密度脂蛋白受体（LDLR）的降解，现在被认为是血浆胆固醇代谢的主要参与者。PCSK 9中的几种功能获得性突变导致高胆固醇血症和过早动脉粥样硬化，因此，抑制PCSK 9诱导的LDLR降解可用于治疗这种致命疾病。在此，我们通过远蛋白印迹、免疫共沉淀和下拉测定发现了内源性PCSK 9结合伴侣。在二维凝胶电泳和质谱分析之后，我们证明了PCSK 9与被鉴定为膜联蛋白A2（AnxA 2）的类似于33 kDa的蛋白质结合，但不与密切相关的膜联蛋白A1结合。此外，我们的功能性LDLR测定和小发夹RNA研究表明，AnxA 2和AnxA2.p11复合物可以阻止HuH 7、HepG 2和中国仓鼠卵巢细胞中PCSK 9介导的LDLR降解。免疫细胞化学显示PCSK 9和AnxA 2共定位于细胞表面，表明可能与LDLR竞争。结构-功能分析表明，PCSK 9的C-末端富含半胱氨酸-组氨酸的结构域与AnxA 2的N-末端重复R1特异性相互作用。对这个70个氨基酸长的重复序列的突变分析表明，AnxA 2的RRTKK 81序列与这种结合有关，因为它突变为AATAA（81）阻止了它与PCSK 9的相互作用。据我们所知，这项工作首次表明PCSK 9对LDLR的活性可以受到内源性抑制剂的调节。AnxA 2的最小抑制序列的鉴定应该为开发用于治疗高胆固醇血症的PCSK 9抑制性先导分子铺平道路。
The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a similar to 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA(81) prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.